The Proteome of Extracellular Vesicles Produced by the Human Gut Bacteria Bacteroides thetaiotaomicron In Vivo Is Influenced by Environmental and Host-Derived Factors.

Bacterial extracellular vesicles (BEVs) released from both Gram-negative and Gram-positive bacteria provide an effective means of communication and trafficking of cell signaling molecules. In the gastrointestinal tract (GIT) BEVs produced by members of the intestinal microbiota can impact host health by mediating microbe-host cell interactions. A major unresolved question, however, is what factors influence the composition of BEV proteins and whether the host influences protein packaging into BEVs and secretion into the GIT. To address this, we have analyzed the proteome of BEVs produced by the major human gut symbiont Bacteroides thetaiotaomicron both in vitro and in vivo in the murine GIT in order to identify proteins specifically enriched in BEVs produced in vivo. We identified 113 proteins enriched in BEVs produced in vivo, the majority (62/113) of which accumulated in BEVs in the absence of any changes in their expression by the parental cells. Among these selectively enriched proteins, we identified dipeptidyl peptidases and an asparaginase and confirmed their increased activity in BEVs produced in vivo. We also showed that intact BEVs are capable of degrading bile acids via a bile salt hydrolase. Collectively these findings provide additional evidence for the dynamic interplay of host-microbe interactions in the GIT and the existence of an active mechanism to drive and enrich a selected group of proteins for secretion into BEVs in the GIT. IMPORTANCE The gastrointestinal tract (GIT) harbors a complex community of microbes termed the microbiota that plays a role in maintaining the host's health and wellbeing. How this comes about and the nature of microbe-host cell interactions in the GIT is still unclear. Recently, nanosized vesicles naturally produced by bacterial constituents of the microbiota have been shown to influence responses of different host cells although the molecular basis and identity of vesicle-born bacterial proteins that mediate these interactions is unclear. We show here that bacterial extracellular vesicles (BEVs) produced by the human symbiont Bacteroides thetaiotaomicron in the GIT are enriched in a set of proteins and enzymes, including dipeptidyl peptidases, an asparaginase and a bile salt hydrolase that can influence host cell biosynthetic pathways. Our results provide new insights into the molecular basis of microbiota-host interactions that are central to maintaining GIT homeostasis and health.

Intestinal Microbiome-Macrophage Crosstalk Contributes to Cholestatic Liver Disease by Promoting Intestinal Permeability in Mice.

BACKGROUND AND AIMS: Mounting evidence supports an association between cholestatic liver disease and changes in the composition of the microbiome. Still, the role of the microbiome in the pathogenesis of this condition remains largely undefined. APPROACH AND RESULTS: To address this, we have used two experimental models, administering alpha-naphtylisocyanate or feeding a 0.1% 3,5-diethoxycarbonyl-1,4-dihydrocollidine diet, to induce cholestatic liver disease in germ-free mice and germ-free mice conventionalized with the microbiome from wild-type, specific pathogen-free animals. Next, we have inhibited macrophage activation by depleting these cells using clodronate liposomes and inhibiting the inflammasome with a specific inhibitor of NOD-, LRR-, and pyrin domain-containing protein 3. Our results demonstrate that cholestasis, the accumulation of bile acids in the liver, fails to promote liver injury in the absence of the microbiome in vivo. Additional in vitro studies supported that endotoxin sensitizes hepatocytes to bile-acid-induced cell death. We also demonstrate that during cholestasis, macrophages contribute to promoting intestinal permeability and to altered microbiome composition through activation of the inflammasome, overall leading to increased endotoxin flux into the cholestatic liver. CONCLUSIONS: We demonstrate that the intestinal microbiome contributes to cholestasis-mediated cell death and inflammation through mechanisms involving activation of the inflammasome in macrophages.

Absence of Bacteria Permits Fungal Gut-To-Brain Translocation and Invasion in Germfree Mice but Ageing Alone Does Not Drive Pathobiont Expansion in Conventionally Raised Mice.

Age-associated changes in the structure of the intestinal microbiome and in its interaction with the brain via the gut-brain axis are increasingly being implicated in neurological and neurodegenerative diseases. Intestinal microbial dysbiosis and translocation of microbes and microbial products including fungal species into the brain have been implicated in the development of dementias such as Alzheimer's disease. Using germ-free mice, we investigated if the fungal gut commensal, Candida albicans, an opportunistic pathogen in humans, can traverse the gastrointestinal barrier and disseminate to brain tissue and whether ageing impacts on the gut mycobiome as a pre-disposing factor in fungal brain infection. C. albicans was detected in different regions of the brain of colonised germ-free mice in both yeast and hyphal cell forms, often in close association with activated (Iba-1+) microglial cells. Using high-throughput ITS1 amplicon sequencing to characterise the faecal gut fungal composition of aged and young SPF mice, we identified several putative gut commensal fungal species with pathobiont potential although their abundance was not significantly different between young and aged mice. Collectively, these results suggest that although some fungal species can travel from the gut to brain where they can induce an inflammatory response, ageing alone is not correlated with significant changes in gut mycobiota composition which could predispose to these events. These results are consistent with a scenario in which significant disruptions to the gut microbiota or intestinal barrier, beyond those which occur with natural ageing, are required to allow fungal escape and brain infection.

The role of the mucin-glycan foraging Ruminococcus gnavus in the communication between the gut and the brain.

Ruminococcus gnavus is a prevalent member of the human gut microbiota, which is over-represented in inflammatory bowel disease and neurological disorders. We previously showed that the ability of R. gnavus to forage on mucins is strain-dependent and associated with sialic acid metabolism. Here, we showed that mice monocolonized with R. gnavus ATCC 29149 (Rg-mice) display changes in major sialic acid derivatives in their cecum content, blood, and brain, which is accompanied by a significant decrease in the percentage of sialylated residues in intestinal mucins relative to germ-free (GF) mice. Changes in metabolites associated with brain function such as tryptamine, indolacetate, and trimethylamine N-oxide were also detected in the cecal content of Rg-mice when compared to GF mice. Next, we investigated the effect of R. gnavus monocolonization on hippocampus cell proliferation and behavior. We observed a significant decrease of PSA-NCAM immunoreactive granule cells in the dentate gyrus (DG) of Rg-mice as compared to GF mice and recruitment of phagocytic microglia in the vicinity. Behavioral assessments suggested an improvement of the spatial working memory in Rg-mice but no change in other cognitive functions. These results were also supported by a significant upregulation of genes involved in proliferation and neuroplasticity. Collectively, these data provide first insights into how R. gnavus metabolites may influence brain regulation and function through modulation of granule cell development and synaptic plasticity in the adult hippocampus. This work has implications for further understanding the mechanisms underpinning the role of R. gnavus in neurological disorders.

Human resident gut microbe Bacteroides thetaiotaomicron regulates colonic neuronal innervation and neurogenic function.

BACKGROUND AND AIMS: As the importance of gut-brain interactions increases, understanding how specific gut microbes interact with the enteric nervous system (ENS), which is the first point of neuronal exposure becomes critical. Our aim was to understand how the dominant human gut bacterium Bacteroides thetaiotaomicron (Bt) regulates anatomical and functional characteristics of the ENS. METHODS: Neuronal cell populations, as well as enteroendocrine cells, were assessed in proximal colonic sections using fluorescent immunohistochemistry in specific pathogen-free (SPF), germ-free (GF) and Bt conventionalized-germ-free mice (Bt-CONV). RNA expression of tight junction proteins and toll-like receptors (TLR) were measured using qPCR. Colonic motility was analyzed using in vitro colonic manometry. RESULTS: Decreased neuronal and vagal afferent innervation observed in GF mice was normalized by Bt-CONV with increased neuronal staining in mucosa and myenteric plexus. Bt-CONV also restored expression of nitric oxide synthase expressing inhibitory neurons and of choline acetyltransferase and substance P expressing excitatory motor neurons comparable to those of SPF mice. Neurite outgrowth and glial cells were upregulated by Bt-CONV. RNA expression of tight junction protein claudin 3 was downregulated while TLR2 was upregulated by Bt-CONV. The enteroendocrine cell subtypes L-cells and enterochromaffin cells were reduced in GF mice, with Bt-CONV restoring L-cell numbers. Motility as measured by colonic migrating motor complexes (CMMCs) increased in GF and Bt-CONV. CONCLUSION: Bt, common gut bacteria, is critical in regulating enteric neuronal and enteroendocrine cell populations, and neurogenic colonic activity. This highlights the potential use of this resident gut bacteria for maintaining healthy gut function.

Regulation of Enteroendocrine Cell Networks by the Major Human Gut Symbiont Bacteroides thetaiotaomicron.

Gut microbes have critical roles in maintaining host physiology, but their effects on epithelial chemosensory enteroendocrine cells (EEC) remain unclear. We investigated the role that the ubiquitous commensal gut bacterium Bacteriodes thetaiotaomicron (Bt) and its major fermentation products, acetate, propionate, and succinate (APS) have in shaping EEC networks in the murine gastrointestinal tract (GIT). The distribution and numbers of EEC populations were assessed in tissues along the GIT by fluorescent immunohistochemistry in specific pathogen free (SPF), germfree (GF) mice, GF mice conventionalized by Bt or Lactobacillus reuteri (Lr), and GF mice administered APS. In parallel, we also assessed the suitability of using intestinal crypt-derived epithelial monolayer cultures for these studies. GF mice up-regulated their EEC network, in terms of a general EEC marker chromogranin A (ChrA) expression, numbers of serotonin-producing enterochromaffin cells, and both hormone-producing K- and L-cells, with a corresponding increase in serum glucagon-like peptide-1 (GLP-1) levels. Bt conventionalization restored EEC numbers to levels in SPF mice with regional specificity; the effects on ChrA and L-cells were mainly in the small intestine, the effects on K-cells and EC cells were most apparent in the colon. By contrast, Lr did not restore EEC networks in conventionalized GF mice. Analysis of secretory epithelial cell monolayer cultures from whole small intestine showed that intestinal monolayers are variable and with the possible exclusion of GIP expressing cells, did not accurately reflect the EEC cell makeup seen in vivo. Regarding the mechanism of action of Bt on EECs, colonization of GF mice with Bt led to the production and accumulation of acetate, propionate and succinate (APS) in the caecum and colon, which when administered at physiological concentrations to GF mice via their drinking water for 10 days mimicked to a large extent the effects of Bt in GF mice. After withdrawal of APS, the changes in some EEC were maintained and, in some cases, were greater than during APS treatment. This data provides evidence of microbiota influences on regulating EEC networks in different regions of the GIT, with a single microbe, Bt, recapitulating its role in a process that may be dependent upon its fermentation products.